Fig. 5

Thr507 phosphorylation of GSR is important for the survival function of AMPKα1. a MS/MS spectra of phosphopeptides containing the phosphotyrosine (pThr) 507 site of GSR. Fragment ions are shown, as is the sequence coverage due to identified fragment ions. All of the highest peaks were explained, but for clarity, they are not all annotated. m/z, mass-to-charge ratio (Upper panel). Thr507 phosphorylation site is highly conserved among human, rat, mouse, and monkey. Identified phosphopeptide are shown above the sequence alignments (Lower panel). b The identified peptide containing the phosphorylated site was shown in two dimension. The phosphorylated site Thr507 and the proton acceptor His511 were indicated in red and green, respectively (Upper panel). Thr507 in the crystal structure of human GSR. Overall view of GSR (Protein Data Bank ID code 3dk8) was shown. The relative positions of His511 (red spheres) and Thr507 (cyan spheres) are indicated (Lower panel). c Immunoblotting of pGSR(T507), total GSR in RKO, and HCT116 cells cultured in glucose deprivation conditions. d GSR enzymatic activities of T/V, T/D, and T/E mutations of Thr507 in HCT116 and RKO cells. e Cell apoptosis of T/V and T/D upon glucose shortage was determined by annexin V/PI staining. f Left: Immunohistochemistry of AMPKα1 and pGSR(T507) in CRC clinical samples. Representative images including corresponding hematoxylin and eosin staining are shown. Right: Scoring of pGSR(T507) in AMPKα1 low and high samples and correlation analysis by using Chi-square test. Results are representative of three experiments. *P < 0.05, **P < 0.01, Student’s t-test, Chi-square test